**4. Discussion**

This study demonstrates that atorvastatin administered in indivudualised doses, tittered to maintain serum LDL cholesterol levels <100mg/dl, significantly decreased lipid profile and oxLDL, reduced carotid artery stenosis in patients managed conservatively and prevented restenosis in patients with prior angioplasty. Oxidised LDL in this study correlated positively with the degree of carotid artery stenosis; it was also shown by multivariate analysis that oxLDL represented an independent risk factor for restenosis. To our knowledge this is the first prospective study with a long observation period of 12 months to report such a clear, significant reduction of oxLDL levels following atorvastatin therapy for carotid atheromatosis of various causes and to report an association of the degree of oxLDL reduction with remission of carotid stenosis. It is also of major importance that this robust, long-standing decline of oxLDL was achieved with doses of atorvastatin used in everyday clinical practice. Interestingly, this beneficial effect was completed in the first six months, while practically no further reduction was noticed past this time point.

The mechanism by which statins modulate oxLDL levels has been controversial in the literature. Moreover, the association of oxLDL level modification with improvement of carotid atheromatosis and clinical outcome is not unequivocally established by large, double-blinded, randomised trials. Under this perspective, the present observational study provides reasona‐ ble evidence that reducing oxLDL may independently improve carotid stenosis.

Carotid intima media thickness (IMT) is a validated measure of carotid atherosclerosis. It is well established that carotid atherosclerosis, serves as an independent surrogate marker for CHD [30] and CVD [31]. Nevertheless, in the present study it was preferred to estimate the degree of carotid stenosis with a more direct approach, because this is more readily available in most hospital settings and because there is an obvious relation with clinical symptoms and signs. Besides, it represents a reliable method with sufficient reproducibility and it is practically the method of choice when evaluated patients candidate for endarterectomy or angioplasty. Evaluating carotid stenosis in turn, is an established method for estimating coronary risk [30] and cardiovascular risk [31]. Other parameters of vessel wall function, such as IMT and plaque morphology, even if clearly associated with cardiovascular risk in the literature, require well equipped laboratory and are not readily available in our hospital. Future research on the field should, ideally, comprise such measurements.

Statins reduce the incidence of cardiovascular events, an effect attributable to their hypocho‐ lesterolemic properties [44]. However, the extent of clinical benefit and accumulating labora‐ tory evidence suggest additional mechanisms of action, the so-called pleiotropic effects [19]. The most important among such effects are the suppression of smooth muscle cell migration and proliferation [45], the reduction of monocyte adhesion to the vascular endothelium [20], the improvement of endothelial function [21], the inhibition of cell-mediated LDL oxidation [22,23], the immuno-modulation of monocyte maturation and differentiation, and the modi‐

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Atorvastatin suppresses cellular uptake of oxLDL from differentiating monocytes by reducing the expression of LOX-1 and scavenger receptors [47] and accelerates the LDL-receptormediated removal of the non oxidized LDL particles [48]. Hydroxymetabolites of atorvastatin protect the LDL against oxidation [31]. The antioxidant potency of atorvastatin metabolites has been confirmed by the reduction of IgG antibodies against LDL, a marker well-associated with CHD [23]. It has even been reported that these active atorvastatin metabolites may have

In acute coronary syndromes, atorvastatin therapy was linked to modulation of short- and long-term immune response towards LDL due to inhibition of lipoprotein-associated phos‐ pholipase A2 (Lp-LPA2) enzyme [34]. The apparent benefit from statin therapy after acute coronary events may also be attributed to the stabilization of the plaque and removal of oxLDL from the vessel wall [50]. Increased mobilization of oxidized phospholipids from the vessel wall, transient binding with apoB-100 particles and clearance from the circulation may be the possible underlying mechanism. Under this perspective the increase in oxLDL:apoB ratio detected with atorvastatin therapy might represent a marker of oxLDL efflux from the vessel wall. Removal of oxLDL contributes to improved endothelial function as oxLDL is highly immunogenic and vasoconstrictive. In our study there was no significant change in oxLDL:apoB ratio. Atorvastatin also inhibits the oxLDL-mediated LOX-1 expression by endothelial cells, the uptake of oxLDL in endothelium and the oxLDL-mediated reduction of protein kinase B (PKB) phosphorylation [24]. The activation of PKB is critical for the expression of eNOS, which promotes vessel relaxation. However, a meta-analysis provided no clear

In STAT trial [52] the antibodies against oxLDL were equally decreased with both aggressive and conventional lipid-lowering therapy. This indicates that the statin-related reduction of oxLDL is not a dose-dependent phenomenon, a finding which is in agreement with our results. It might therefore represent a pleiotropic effect, independent -at least partially- from the hypocholesterolemic action. A study by Orem et al detected a significant decrease of autoantibodies against oxLDL with low doses of atorvastatin (10mg) [53], similar to doses used in our study. In statin exposed patients, intensification of the regimen offers no additional benefit and only those with LDL>125mg/dl benefited from a more aggressive statin therapy [52]. Statins have a dose-related response with regard to clinical outcome, but this dose-related response has not been confirmed with regard to oxidative stress [54]. This might alternatively be explained by the hypothesis that statins achieve their uttermost benefit on oxLDL after a certain time point [52], after which further continuation of treatment serves only the purpose of maintenance.

fication of production of inflammatory cytokines [46].

greater anti-atherosclerotic effects than other statin molecules [49].

evidence that statin therapy have a favourable effect on oxLDL [51].

hoxLDL=0: low quartile of oxidised LDL levels, hoxLDL=1: high quartile of oxidised LDL levels, 0=baseline, 1=one month, 2=three months, 3=six months, 4=twelve months.

**Figure 7.** Kaplan Meier survival analysis for the estimation of the risk ratio for restenosis according to the levels of oxidised LDL (oxLDL). With red line those with oxLDL levels in the highest quartile of the values. With blue line those with oxLDL levels in the lowest quartile of the measurements (risk ratio 1.025, logrank test p<0.001).

Oxidised LDL has long been recognized as a risk factor for carotid atherosclerosis in asymp‐ tomatic men [32] and has also been linked with CVD [33]. Oxidized LDL levels [34], autoan‐ tibodies against epitopes of oxLDL [34] and oxLDL:LDL ratio [30] are independently associated with increased risk for coronary atheromatosis and ischemic heart disease. In‐ creased levels of oxLDL [9] and MDA-LDL [10] in such cases are related to plaque instability. On the other hand, it has been reported that oxLDL is weakly associated with carotid IMT, but not with carotid plaque occurrence [35]. Oxidised LDL impairs endothelium relaxation [36] by inhibition of the expression of eNOS and of the transport pathways of nitric oxide (NO) from the endothelial cell, reduces the responsiveness of smooth muscle cell to NO [37], inhibits the NO-mediated vasodilation [16,36,38], induces the expression of adhesion molecules [39], acts directly chemotactic to circulating monocytes [16], stimulates endothelial cells to produce monocyte chemoattractant protein-1 (MCP-1) [40], facilitates monocyte adhesion to intima [41], exhibits cytotoxic properties against endothelial cells [16], and induces the expression of inflammatory molecules [16]. All of the above contribute directly to dysfunction of the endothelium [13] and foam cell formation, which is the first step in the development of fatty streaks [18], the first visible step of atherosclerosis. These effects are mediated by preferential binding of oxLDL with type A scavenger receptors (SRA, SRA-II and CD36) on subendothelial resident macrophages and smooth muscle cells [42] and lectin-like oxLDL receptor-1 (LOX-1) on endothelial cells [43] rather than the typical LDL receptor, resulting in an unrestricted uptake of cholesterol.

Statins reduce the incidence of cardiovascular events, an effect attributable to their hypocho‐ lesterolemic properties [44]. However, the extent of clinical benefit and accumulating labora‐ tory evidence suggest additional mechanisms of action, the so-called pleiotropic effects [19]. The most important among such effects are the suppression of smooth muscle cell migration and proliferation [45], the reduction of monocyte adhesion to the vascular endothelium [20], the improvement of endothelial function [21], the inhibition of cell-mediated LDL oxidation [22,23], the immuno-modulation of monocyte maturation and differentiation, and the modi‐ fication of production of inflammatory cytokines [46].

Atorvastatin suppresses cellular uptake of oxLDL from differentiating monocytes by reducing the expression of LOX-1 and scavenger receptors [47] and accelerates the LDL-receptormediated removal of the non oxidized LDL particles [48]. Hydroxymetabolites of atorvastatin protect the LDL against oxidation [31]. The antioxidant potency of atorvastatin metabolites has been confirmed by the reduction of IgG antibodies against LDL, a marker well-associated with CHD [23]. It has even been reported that these active atorvastatin metabolites may have greater anti-atherosclerotic effects than other statin molecules [49].

In acute coronary syndromes, atorvastatin therapy was linked to modulation of short- and long-term immune response towards LDL due to inhibition of lipoprotein-associated phos‐ pholipase A2 (Lp-LPA2) enzyme [34]. The apparent benefit from statin therapy after acute coronary events may also be attributed to the stabilization of the plaque and removal of oxLDL from the vessel wall [50]. Increased mobilization of oxidized phospholipids from the vessel wall, transient binding with apoB-100 particles and clearance from the circulation may be the possible underlying mechanism. Under this perspective the increase in oxLDL:apoB ratio detected with atorvastatin therapy might represent a marker of oxLDL efflux from the vessel wall. Removal of oxLDL contributes to improved endothelial function as oxLDL is highly immunogenic and vasoconstrictive. In our study there was no significant change in oxLDL:apoB ratio. Atorvastatin also inhibits the oxLDL-mediated LOX-1 expression by endothelial cells, the uptake of oxLDL in endothelium and the oxLDL-mediated reduction of protein kinase B (PKB) phosphorylation [24]. The activation of PKB is critical for the expression of eNOS, which promotes vessel relaxation. However, a meta-analysis provided no clear evidence that statin therapy have a favourable effect on oxLDL [51].

Oxidised LDL has long been recognized as a risk factor for carotid atherosclerosis in asymp‐ tomatic men [32] and has also been linked with CVD [33]. Oxidized LDL levels [34], autoan‐ tibodies against epitopes of oxLDL [34] and oxLDL:LDL ratio [30] are independently associated with increased risk for coronary atheromatosis and ischemic heart disease. In‐ creased levels of oxLDL [9] and MDA-LDL [10] in such cases are related to plaque instability. On the other hand, it has been reported that oxLDL is weakly associated with carotid IMT, but not with carotid plaque occurrence [35]. Oxidised LDL impairs endothelium relaxation [36] by inhibition of the expression of eNOS and of the transport pathways of nitric oxide (NO) from the endothelial cell, reduces the responsiveness of smooth muscle cell to NO [37], inhibits the NO-mediated vasodilation [16,36,38], induces the expression of adhesion molecules [39], acts directly chemotactic to circulating monocytes [16], stimulates endothelial cells to produce monocyte chemoattractant protein-1 (MCP-1) [40], facilitates monocyte adhesion to intima [41], exhibits cytotoxic properties against endothelial cells [16], and induces the expression of inflammatory molecules [16]. All of the above contribute directly to dysfunction of the endothelium [13] and foam cell formation, which is the first step in the development of fatty streaks [18], the first visible step of atherosclerosis. These effects are mediated by preferential binding of oxLDL with type A scavenger receptors (SRA, SRA-II and CD36) on subendothelial resident macrophages and smooth muscle cells [42] and lectin-like oxLDL receptor-1 (LOX-1) on endothelial cells [43] rather than the typical LDL receptor, resulting in an unrestricted

hoxLDL=0: low quartile of oxidised LDL levels, hoxLDL=1: high quartile of oxidised LDL levels, 0=baseline, 1=one

**Figure 7.** Kaplan Meier survival analysis for the estimation of the risk ratio for restenosis according to the levels of oxidised LDL (oxLDL). With red line those with oxLDL levels in the highest quartile of the values. With blue line those

with oxLDL levels in the lowest quartile of the measurements (risk ratio 1.025, logrank test p<0.001).

0 1 2 3 4 5 Time of observation (in five periods)

Kaplan Meier Curves for Carotid Stenosis

hoxHDL = 0 hoxHDL = 1

Logrank test p<0.001

uptake of cholesterol.

0.00

month, 2=three months, 3=six months, 4=twelve months.

0.25

0.50

Carotid Stenosis %

0.75

1.00

138 Carotid Artery Disease - From Bench to Bedside and Beyond

In STAT trial [52] the antibodies against oxLDL were equally decreased with both aggressive and conventional lipid-lowering therapy. This indicates that the statin-related reduction of oxLDL is not a dose-dependent phenomenon, a finding which is in agreement with our results. It might therefore represent a pleiotropic effect, independent -at least partially- from the hypocholesterolemic action. A study by Orem et al detected a significant decrease of autoantibodies against oxLDL with low doses of atorvastatin (10mg) [53], similar to doses used in our study. In statin exposed patients, intensification of the regimen offers no additional benefit and only those with LDL>125mg/dl benefited from a more aggressive statin therapy [52]. Statins have a dose-related response with regard to clinical outcome, but this dose-related response has not been confirmed with regard to oxidative stress [54]. This might alternatively be explained by the hypothesis that statins achieve their uttermost benefit on oxLDL after a certain time point [52], after which further continuation of treatment serves only the purpose of maintenance. Atorvastatin has been shown to reduce small dense LDL subfractions, remnant-like particles cholesterol and oxLDL, and improve endothelial function, after just few weeks of therapy [55,56]. Such time-related effect has not been fully elucidated, but may possibly account for our finding that in the first six months there was an accelerated decline of oxLDL levels followed by a milder reduction rate thereafter.

possibly counteract the oxLDL-associated increase of NF-κΒ, and therefore, the production of such cell adhesion molecules [60]. Statins also enhance scavenger receptor expression in macrophages [60], and increase plaque stability via reduction of metalloproteinases [60].

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The reduction of oxidised LDL and of carotid stenosis in our study was relevant for both, smokers and non-smokers. However, during subgroup analysis showed that the beneficial effect of statin use concerns mostly the subgroup of mild smokers, while no such effect was noticed for moderate and heavy smokers. How smoking may diminish the beneficial effect of statins on oxidized LDL and carotid stenosis is not yet clarified in the literature. A reasonable assumption might be that, since smoking increases the oxidative stress, it contributes to enhanced LDL oxidation [63]. Moreover, studies in animal models, have demonstrated that smoking alters the immunologic response to oxidized LDL by reducing the production of antibodies against these molecules, i.e. causing a kind of immune suppression regarding the

The Mercodia oxLDL detects the MDA-modified apoB [28]. It has been proposed that oxLDL looses its predictive value for CVD when adjustment for apoB level is performed [54]. In several studies though, a significant reduction of Mercodia oxLDL with atorvastatin 10mg was still detected even after adjustment for apoB, [10,31,54], while in other studies no adjustment for LDL or apoB levels was made [54,65]. In our study the oxLDL:apoB ratio remained unchanged, but in the multivariate analysis the reduction of oxLDL was still significant after adjustment

In patients with familial hypercholesterolemia a lack of association between oxLDL and IMT was reported at baseline, however two years therapy with atorvastatin 80mg was associated with regression of carotid IMT [66]. The LDL subfraction profile and autoantibodies against oxLDL remained unchanged. Nevertheless, the rate of oxidation and the amount of dienes formed decreased and this was linked to lessening of atherosclerosis. In our study the reduction of carotid stenosis was associated with decreased oxLDL levels. Besides, the unchanged oxLDL autoantibodies levels do not preclude the reduction of oxLDL, as was indicated in another study involving dialysis patients, where atorvastatin therapy reduced

Disadvantages of the study were the relatively small size, the lack of a control group com‐ prising of patients with carotid stenosis not on statin therapy, which would be unethical, the fact that researchers were not blinded to the patients' status, the lack of randomization of the

This prospective, cross-sectional study with such a long observation period provided enough evidence to postulate a favourable effect of low-dose atorvastatin therapy on oxLDL, which was additionally associated with improvement of stenosis in patients with carotid atheroma‐ tosis. We thus, assume that oxidised LDL may represent a far more sensitive risk factor for

plasma oxLDL, whereas oxLDL autoantibodies did not changed significantly [67].

dose-schedules and the use of only one method to detect oxLDL.

response to oxidized LDL. Thus, it has been shown to increase carotid IMT [64].

for apoB and LDL levels.

**5. Conclusion**

Additional pleiotropic effects of statins have been reported in the literature and might account for the observed beneficial effects in the current study. Lysophosphatidylcholine is elevated during LDL oxidation and is responsible for some of the biological effects of oxLDL. Atorvas‐ tatin alters the ability of oxLDL to impair the endothelium relaxation, by modulating the hydrolysis of phosphatidylcholine to lysophosphatidylcholine when LDL is being oxidized [57]. Statins remove predominately "aged LDL" from plasma, which is more prone to oxidation [53], through stimulation of hepatic LDL receptor activity and inhibition of very-low density lipoprotein (VLDL) and LDL production by the liver cells [53]. Statins also reduce oxygen species generation [54]. Atorvastatin promotes adipocyte uptake of oxLDL in rabbits by increasing the expression of CD36 and peroxisome proliferators-activated receptor γ (PPARγ) in adipocytes [58]. The increased expression of such receptors by adipocytes results to internalization of oxLDL and clearance from plasma, converting adipocytes to an oxLDLbuffering pool [58]. Reduction of oxLDL in patients with CHD with atorvastatin 10mg parallel with an increase of adiponectin, which has anti-atherogenic [55], anti-inflammatory and antidiabetic [55] properties through reduction of insulin resistance [55]. The CARDS study reported a significant degree of preventive activity of atorvastatin against myocardial infarc‐ tion in eucholesterolemic diabetic patients, conceivably attributed to such improvement of insulin sensitivity [55]. Statins also diminish the expression of CD40 and CD40 ligand in vascular cells, smooth muscle cells and macrophages, which are promoted by oxLDL and are considered proatherogenic [59]. Other anti-inflammatory pathways include reduction of Creactive protein [60], chemokines, major histocompatibility complex II molecules, matrixdegrading enzymes, and procoagulant tissue factor [59]. Atorvastatin reverses the oxLDLmediated inhibition of vascular endothelial growth factor-induced endothelial progenitor cell differentiation via the phosphatidylinositol 3 kinase/Akt pathway [61], which restores the oxLDL-related inhibition of mature endothelial cells migration [61]. This could improve neovascularization and collateral vessel formation in response to tissue ischemia. Atorvastatin also suppresses platelet activity [62] by reducing the expression of CD36 and LOX-1, which are present in platelets [43,62], thus inhibiting the oxLDL-mediated platelet hyperactivity [62]. Statins reduce the oxLDL-derived expression of adhesion molecules (E- and P-selectins, vascular cell adhesion molecule 1 [VCAM-1] and intercellular adhesion molecule 1 [ICAM-1]) in human coronary artery endothelial cells [15], through up-regulation of eNOS expression [15], which regulates the expression of adhesion molecules in endothelial cells [15]. Statins also diminish the oxLDL-mediated activation of nuclear factor-κΒ (NF-κB) [15], which regulates the transcription of adhesion molecule genes [33]. In diabetic patients with dyslipidemia atorvastatin reduced CVD and markers of inflammation, adhesion and oxidation, such as CRP, soluble ICAM-1, soluble VCAM-1, E-selectin, matrix metalloproteinase 9, secretory phospho‐ lipase A2 (sPLA2), and oxLDL, the latter by 38,4% [60]. Moreover, the change of oxLDL levels correlated with the change of sICAM-1 and E-selectin levels, suggesting that statins could possibly counteract the oxLDL-associated increase of NF-κΒ, and therefore, the production of such cell adhesion molecules [60]. Statins also enhance scavenger receptor expression in macrophages [60], and increase plaque stability via reduction of metalloproteinases [60].

The reduction of oxidised LDL and of carotid stenosis in our study was relevant for both, smokers and non-smokers. However, during subgroup analysis showed that the beneficial effect of statin use concerns mostly the subgroup of mild smokers, while no such effect was noticed for moderate and heavy smokers. How smoking may diminish the beneficial effect of statins on oxidized LDL and carotid stenosis is not yet clarified in the literature. A reasonable assumption might be that, since smoking increases the oxidative stress, it contributes to enhanced LDL oxidation [63]. Moreover, studies in animal models, have demonstrated that smoking alters the immunologic response to oxidized LDL by reducing the production of antibodies against these molecules, i.e. causing a kind of immune suppression regarding the response to oxidized LDL. Thus, it has been shown to increase carotid IMT [64].

The Mercodia oxLDL detects the MDA-modified apoB [28]. It has been proposed that oxLDL looses its predictive value for CVD when adjustment for apoB level is performed [54]. In several studies though, a significant reduction of Mercodia oxLDL with atorvastatin 10mg was still detected even after adjustment for apoB, [10,31,54], while in other studies no adjustment for LDL or apoB levels was made [54,65]. In our study the oxLDL:apoB ratio remained unchanged, but in the multivariate analysis the reduction of oxLDL was still significant after adjustment for apoB and LDL levels.

In patients with familial hypercholesterolemia a lack of association between oxLDL and IMT was reported at baseline, however two years therapy with atorvastatin 80mg was associated with regression of carotid IMT [66]. The LDL subfraction profile and autoantibodies against oxLDL remained unchanged. Nevertheless, the rate of oxidation and the amount of dienes formed decreased and this was linked to lessening of atherosclerosis. In our study the reduction of carotid stenosis was associated with decreased oxLDL levels. Besides, the unchanged oxLDL autoantibodies levels do not preclude the reduction of oxLDL, as was indicated in another study involving dialysis patients, where atorvastatin therapy reduced plasma oxLDL, whereas oxLDL autoantibodies did not changed significantly [67].

Disadvantages of the study were the relatively small size, the lack of a control group com‐ prising of patients with carotid stenosis not on statin therapy, which would be unethical, the fact that researchers were not blinded to the patients' status, the lack of randomization of the dose-schedules and the use of only one method to detect oxLDL.
